Figure 5

CHD4 chromatin association is enhanced following DNA damage. (A) HeLa cells we treated with 6Gy IR or untreated, 1 hr following IR treatment the cells chromatin fractionation analysis was performed. Whole cell extract (WCE) and chromatin fraction lysates were then analysed by western blot analysis with CHD4 as indicated. (B) ATM proficient (C3ABR) and ATM deficient (L3) cells we treated with 6Gy IR or untreated, 1 hr following IR treatment the cells chromatin fractionation analysis was performed. Whole cell extract (WCE) and chromatin fraction lysates were then analysed by western blot analysis with CHD4. (C) HeLa cells were either untransfected or transfected with plasmid DNA encoding Flag-CHD4 or Flag-CHD4 S1349A (CHD4-A). 24 hr after transfection the cells were treated with 6Gy IR. Cells were harvested at the times indicated and chromatin fractionation analysis was performed. WCE and chromatin fraction lysates were then subjected to western blot analysis with CHD4. (D, E) U2OS cells were either mock transfected or transfected with siCHD4 or with plasmid DNA encoding Flag-CHD4 or Flag-CHD4 S1349A (CHD4-A), 36 hr after transfection cells were detergent pre-extracted to remove non-chromatin bound proteins prior to fixation. Immunofluorescence analysis for 53BP1 (D) and (E) γH2AX was then performed and the percentage of cells (+ and - standard deviation) with multiple foci was calculated from a count of at least 100 cells across 3 independent experiments. (F) U2OS cells were transfected with plasmid DNA encoding Flag-CHD4 (CHD4) or Flag-CHD4 S1349A (CHD4-A), 24 hr after transfection cells were treated with 2Gy IR. Prior to IR and 1 hr and 6 hr after IR, cells were detergent pre-extracted to remove non-chromatin bound proteins prior then fixed for immunofluorescence analysis. The percentage of γH2AX positive cells (+ and - standard deviation) was then calculated from a count of at least 100 cells in 3 independent experiments.